Directed evolution of Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles

Artificial metalloenzymes (ArMs) are constructed by anchoring organometallic catalysts to an evolvable protein scaffold. They present the advantages of both components and exhibit considerable potential for the in vivo catalysis of new-to-nature reactions. Herein, Escherichia coli surface-displayed Vitreoscilla hemoglobin (VHbSD-Co) that anchored the cobalt porphyrin cofactor instead of the original heme cofactor was used as an artificial thiourea oxidase (ATOase) to synthesize 5-imino-1,2,4-thiadiazoles. After two rounds of directed evolution using combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy, the evolved six-site mutation VHbSD-Co (6SM-VHbSD-Co) exhibited significant improvement in catalytic activity, with a broad substrate scope (31 examples) and high yields with whole cells. This study shows the potential of using VHb ArMs in new-to-nature reactions and demonstrates the applicability of E. coli surface-displayed methods to enhance catalytic properties through the substitution of porphyrin cofactors in hemoproteins in vivo.


EDGE ARTICLE
EtOH : H 2 O (4 : 1) at room temperature (Scheme 1e). 11However, some drawbacks exist, such as the use of high temperature, harmful organic solvents, or the limitation of the substrate scope.8][29][30][31][32][33] Our group previously reported a Co(ppIX)linked ArM that was designed based on Vitreoscilla hemoglobin (VHb). 34,35The cobalt porphyrin cofactor was incorporated into the cavity of VHb (VHb Co ) in vivo by using a porphyrin synthesis-decient Escherichia coli strain RP523, 36 and a range of engineered VHb Co variants were found to catalyze intramolecular oxidative cyclization for the synthesis of 2-substituted benzoxazoles/benzothiazoles.However, many metal cofactors are inhibited by cellular components and have limited access to the cytoplasm, indicating that the scaffold protein must be puried. 37These factors limit the throughput of genetic optimization schemes applied to ArMs and the applicability in vivo to expand natural metabolism.In recent decades, numerous studies have focused on making the assembly and screening of these biohybrid catalysts more convenient: (i) in vivo assembly of ArM with biosynthesized cobalt protoporphyrin IX (Co(ppIX)) under ironlimited, cobalt-rich growth conditions, 38,39 (ii) in vivo assembly of ArM and cofactor with a system to transport the cofactor into the cytoplasm (e.g., recombinant production of heme proteins in E. coli strain Nissle 1917, 40,41 ChuA 42 and the Hug 43 system for porphyrin transportation), and (iii) in vivo screening via cell surface display. 37,44,45o circumvent the limitation of the directed evolution of VHb Co based on our previous studies, 34,35,46,47 we developed a new approach that uses E. coli surface-displayed method.We created a platform to display VHb Co on E. coli's outer membrane (VHb SD-Co ) and established a whole-cell high-throughput screening strategy based on ultraviolet (UV) absorption for the in vivo directed evolution of VHb SD-Co with high catalytic reactivity for the synthesis of 5-imino-1,2,4-thiadiazoles.Aer two rounds of evolution, the best VHb SD-Co mutant exhibited excellent activity, producing thiadiazole products with broad substrate scopes, and it can be further evolved in different directions by adjusting the workow.Our study provides a case for the systematic implementation and directed evolution of an ArM that can be applied to an in vivo non-natural reaction by using O 2 as the oxidant and H 2 O as the solvent.Notably, this study is the rst successful attempt to catalyze the synthesis of 1,2,4-thiadiazoles by using a biocatalyst, and it exhibits considerable potential for the further exploration of other nonnatural reactions in vivo.

Result and discussion
Our study started with the reaction of benzamidine (1a) and phenyl isothiocyanate (2a) under aerobic conditions at room temperature to form the 5-imino-1,2,4-thiadiazole compound 3aa catalyzed by wild-type VHb.Apart from VHb, several hemeproteins and ArMs that were reported in our previous works were also evaluated (Table 1). 34,35The synthesis of 3aa could not proceed without a catalyst, as shown in entry 1. Cytochrome C, myoglobin, and horseradish peroxidase exhibited low reactivity, while hemoglobins catalyzed the formation of 3aa with better yields (entries 2-10).Among all of these hemeproteins, VHb presented the highest reactivity.Similar to the oxidation reactions reported in previous works, VHb Co demonstrated promising activity compared with wild-type VHb and VHbArM with Mn(ppIX) (VHb Mn ) and Zn(ppIX) (VHb Zn ) (entries 11-13).Therefore, VHb Co was selected to be the best biocatalyst for further study.
Considering that the intracellular assembly of VHb Co is inefficient for mutant screening and larger substrates experience difficulty in entering E. coli cells, we developed a VHb Co surface display platform on the outer membrane of E. coli by using Lpp-OmpA anchor.By fusing truncated E. coli lipoprotein Lpp, the rst ve b-strands of outer membrane protein OmpA, a Gly×5 linker, and VHb protein (with His×6 tag on the Cterminus), VHb was anchored onto the outer membrane.To demonstrate the successful construction of Lpp-OmpA-VHb fusion ArM (VHb SD-Co ), E. coli cells were stained with a primary mouse-anti his-tag antibody aer expression and labeled with a secondary uorescent antibody, followed by uorescence microscopy analysis (Fig. 1).Antibodies could not easily cross the membrane and stain the VHb Co protein expressed in the cytoplasm as we anticipated (Fig. 1b).The control experiment of E. coli cells without VHb protein revealed the uorescence labeling of E. coli only in the presence of His×6 tag-labeled VHb displayed on the surface of the outer membrane (Fig. 1a and c).As expected, these results were consistent with previous reports of Lpp-OmpA-labeled proteins. 37,44,45,48,49o test the catalytic activity of VHb SD-Co , the fusion protein was expressed in E. coli cells under anaerobic conditions.The cells were spun-down, and the pellets were resuspended in substrate buffer (1a 50 mM, 2a 50 mM, and K 2 CO 3 30 mM).Then, the reaction mixture was incubated at room temperature for 12 h.The yield of the reaction was determined via highperformance liquid chromatography.As indicated in Table 2, puried fusion protein (entry 1) and cell lysis (entry 2) exhibited similar performance to VHb Co .The reaction catalyzed by VHb SD- Co on the whole cells demonstrated a noticeable enhancement in yield and turnover numbers (TON) (entry 3).To further demonstrate the feasibility of VHb SD-Co catalyzed in whole cells, a series of optimization experiments was carried out (Tables S1 † and 2), and the results indicated that VHb SD scaffold, Co(ppIX) cofactor, O 2 , K 2 CO 3 (for the neutralization of HCl in amidine salt), and aqueous solvent played essential roles in this biocatalysis method.The optimized conditions that led to a slight increase in yield and TON of 3aa (71%, 1420 TON) are as follows: when O 2 is used as oxidant under aerobic conditions, VHb SD-Co catalyzes the synthesis of 3aa from 1a (50 mM) and 2a (50 mM) with K 2 CO 3 (30 mM) as base in phosphate-buffered solution (PBS) (5% MeCN for the solubilization of substrates) for 16 h.
We also tested several previously reported VHb Co variants and mutants of axial His residue (Table S2 †), but they did not perform better than the wild-type VHb Co due to the different steric hindrance effects of substrates.Therefore, we developed a high-throughput screening method to select VHb SD-Co variants as articial thiourea oxidase (ATOase) with better catalytic activity.This method utilized the UV absorption discrepancy of N-(phenylcarbamothioyl)benzimidamide intermediate 4aa and product 3aa (Fig. S1 † and 2a).Intermediate 4aa presented a characteristic absorption peak at 313 nm, while product 3aa had no UV absorption at this wavelength.In accordance with previously reported studies, 50 substrates 1a and 2a formed intermediate 4aa spontaneously, enabling us to screen the VHb SD-Co variants based on the decrease in UV absorption with reaction in 96-well plates (Fig. 2b).
To identify potential mutation sites, intermediate 4aa was docked into the structure of VHb SD-Co (with Co III -superoxide intermediate) by using Autodock.The docking result indicated that Y29 residue on the B helix, and F43, Q53, P54, K55, and L57 residues on the loose loop region were closely related to the access of 4aa.Among them, Y29 was directly above the porphyrin plane and limited the exposure of intermediate 4aa to Co III -superoxide intermediate.Therefore, a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy 51,52 was applied to evolve this ATOase, the six residues were divided into two groups (Y29 and F43/Q53/   P54/K55/L57) according to the region they located.At rst, a site saturation mutagenesis (SSM) library at Y29 was generated, and VHb SD-Co mutants performed signicant changes compared with WT VHb SD-Co .Three better VHb SD-Co single mutants were identied in the surface-displayed whole-cell screening and the yield was reevaluated separately (yield of 3aa: VHb SD-Co Y29G, 81%; VHb SD-Co Y29A, 77%; VHb SD-Co Y29F, 76%).On the basis of the best single-site mutation VHb SD-Co Y29G (dened as 1SM-VHb SD-Co ), a CAST library of F43, Q53, P54, K55, and L57 residues was created.Aer this high-throughput screening, several hits were identied (Table S3 †) and the best six-site mutation, VHb SD-Co Y29G-F43P-Q53P-P54G-K55L-L57A (dened as 6SM-VHb SD-Co ), was reevaluated and afforded an improved activity (yield of 3aa: 93%), and the SDS-PAGE verication was showed in Fig. S2.† The substrate scope of amidines (1) and isothiocyanates (2) was explored using the best ATOase (6SM-VHb SD-Co ), and the generality of this biocatalytic method for the synthesis of thiadiazoles was investigated.The results indicated that this biocatalyst was compatible with a range of amidines and isothiocyanates under standard conditions.As shown in Table 3, both electron-donating groups and electronwithdrawing groups on the phenyl groups of amidines (1) were applicable to the 6SM-VHb SD-Co (3ba-3la) and afforded excellent yields (85-96%).Benzamidine with a large spatialresistance group showed a decrease on the reactivity, it might because the biphenyl benzamidine is a big substrate which is harder to access into the cavity of VHb, and also probably dispersed into the membrane due to the less water solubility (3ha).Meanwhile, small substituents on the phenyl group of substrates 1 do not lead to an increase in reactivity.This phenomenon may be due to the imperfect matching of intermediate 4 and the active cavity providing the worse catalytic performance based on the neighborhood and orientation effects of the enzyme.The yield of product 3ma-3oa presented varying enhancement by replacing 6SM-VHb SD-Co with 1SM-VHb SD-Co (the mutant with smaller substrate cavity).
Thereaer, the scope of isothiocyanates was investigated and demonstrated a similar pattern to amidines (Table 4).The steric effects inuenced the overall yield of thiadiazoles as demonstrated by the ortho-and para-substituted phenyl isothiocyanates producing decreased yields of products, compared with their meta-substituted counterparts (3ca, 3ia, and 3ja; 3ea, 3ka, and 3la).Encouraged by the successful results, we focused on the further application of this evolved ATOase.A preparation-scale reaction (0.5 mmol) for product 3aa was performed and afforded a satisfactory yield (95%).
To clarify the mechanism of this reaction, some control experiments were performed under specic conditions (Table 5).The results indicated that without ATOase 6SM-VHb SD-Co , substrates 1a and 2a coupled and converted into intermediate 4aa spontaneously (entry 1).With the addition of ATOase, intermediate 4aa afforded 3aa in 98% yield (entry 2).When the reaction was conducted in the presence of radical scavenger 2,2,6,6-tetramethylpiperdine-1-oxide (TEMPO), the yield of 3aa decreased dramatically, implying the involvement of a radical pathway in this ATOase-catalyzed reaction (entry 3).

Conclusions
We evolved an articial thiourea oxidase based on the porphyrin substitution of VHb.The oxidase catalyzed amidines (1) and isothiocyanates (2) to form 5-imino-1,2,4-thiadiazoles (3).To simplify the protocol of ArM construction and the screening of directed evolution, we implemented an E. coli surface-displayed method by using Lpp-OmpA-VHb fusion protein.Through a CAST/ISM evolution of Y29, F43, Q53, P54, K55, and L57 residues, we obtained a six-site mutation 6SM-VHb SD-Co (Y29G-F43P-Q53P-P54G-K55L-L57A) with improved ATOase activity (up to 96% yield and 1920 TON, 2.5-fold increased versus WT VHb) and broad substrate scopes (31  examples).The molecular docking of ATOase and thiourea intermediate 4 further indicated the rationale of the reactivity enhancement.We expect that this surface-displayed method can be extended to streamline the construction and directed evolution of ArMs for new-to-nature reactions and in vivo synthetic biology explorations.

Data availability
All the data supporting this article have been included in ESI.†

Fig. 1
Fig. 1 Fluorescence microscopy of immuno-stained E. coli cells.(a) E. coli cells without VHb, (b) E. coli cells expressing VHb Co in the cytoplasm, (c) E. coli cells expressing VHb SD-Co on the surface.Cells were labeled with a primary mouse anti-6xhistag-antibody followed by a fluorescently-labeled secondary goat-anti-mouse antibody.

The control experiments entries 4
and 5 demonstrated the essential role of the O 2 and Co(ppIX) cofactor of ATOase.On the basis of the previous literature 10,53,54 and our earlier study on VHb Co -catalyzed aerobic oxidation,34,35 the mechanism of this reaction was proposed as shown in Scheme 2. First, amidines (1) and isothiocyanates (2) were coupled spontaneously to generate thiourea intermediate 4.Under aerobic conditions, the Co(ppIX) cofactor interacted with O 2 to generate a Co III -superoxide intermediate, which acidied 4 to form a thiyl radical I through an electron transfer process and converted to a base itself, and then thiyl radical I transformed to intermediate II aer proton transfer.Next, the nucleophilic attack on the sulfur atom by imino nitrogen led to cyclization to form intermediate III, which was accompanied by a proton transfer to Co III -superoxide intermediate and generated a peroxo anion.Aer proton exchange with solvent, III converted into 5-imino-1,2,4-thiadiazole product 3 and H 2 O 2 was generated simultaneously which could also facilitate this reaction (the generation of H 2 O 2 was shown in Fig. S3 †).
depicts the interactions of intermediate 4aa with VHb SD-Co , 1SM-VHb SD-Co and 6SM-VHb SD-Co (the structure of 1SM-VHb SD-Co and 6SM-VHb SD-Co were constructed by homology modeling strategy using SWISS-MODEL 57 ).In particular, intermediate 4aa was accessed in a hydrophobic pocket in 6SM-VHb SD-Co and well-accommodated into the active center by van der Waals contacts between proximate residues (F28, G29, F33, P43, D44, L55 and A58) and the thiourea intermediate, residue G54 stabilized intermediate by forming a hydrogen bond with the imino nitrogen on the intermediate, meanwhile, pi-alkyl interactions (L32, A57 and V98) and amide-pi stacked interactions (P53) also contributes to the approach of the intermediate (a 2D diagram showed by Discovery Studio Visualizer 4.0 (ref.58) in Fig. S4 †).The sulfur atom on intermediate 4aa maintains a slightly far distance from the oxygen atom on Co III -superoxide intermediate in WT-VHb SD-Co (Fig. 3a), and the mutation of six amino acids with shorter side chains (Gly, Ala) and rigid side chains (Pro) adjusts the conformation of the exible loop region (CD spectrum of WT-VHb SD-Co and 6SM-VHb SD-Co were showed in Fig. S5 †), reduces spatial resistance to the intermediate, and brings the distance between intermediate 4aa and the oxygen atom in the Co III -superoxide intermediate closer in mutant 6SM-VHb SD-Co (Fig. 3c).

Fig. 3
Fig. 3 Structure model of thiourea intermediate 4aa (light purple) in the active site of VHb SD-Co .(a) Structure of WT-VHb SD-Co in complex with 4aa.(b) Structure of 1SM-VHb SD-Co in complex with 4aa.(c) Structure of 6SM-VHb SD-Co in complex with 4aa.